Detection and quantification of differentially expressed genes in a number of pathological conditions such as different benign and malignant tumors, neurological disorders, heart disease and autoimmune disorders, would be useful in the diagnosis, prognosis and treatment of these pathological conditions. Quantification of gene expression would also be useful in diagnosis of infectious diseases and following up effects of pharmaceuticals or toxins on molecular level. For example, gene expression data could be used to determine the pharmacological mechanism of a drug or a toxin (Libutti et al., Microarray technology and gene expression analysis for the study of angiogenesis. Expert Opin Biol Ther. 2002 June; 2(5):545-56).
The methods for transcript detection and quantification have traditionally included Northern-blot hybridization, ribonuclease protection assay, and reverse transcriptase polymerase chain reaction (RT-PCR) based methods. However, in addition to suffering from lack of sensitivity (except RT-PCR), these methods are only useful for roughly estimating the relative expression changes of each transcript among samples from different sources. The different RT-PCR based techniques are the most suitable quantification method for diagnostic purposes, because they are very sensitive and thus require only a small sample size which is desirable for a diagnostic test.
Absolute quantification of transcript copy numbers in a sample is a requirement if one wishes to compare gene expression between samples and even within the same sample. However, quantification of nucleic acid copy numbers is difficult using PCR based methods because of the inherent non-linear nature of the PCR reaction. PCR amplification will change from an exponential phase to a plateau phase with the consumption of reagents or enzyme inactivation. Often, the exponential phase of the PCR must be determined separately which may involve sampling of the PCR reactions at different time points or performing the PCR using different dilutions of the template. Further, because of differences in amplification efficiency between templates, the starting quantities of different PCR products cannot be compared directly even in the linear range. Detection of PCR products has traditionally been performed after amplification is completed. Typically, an aliquot of the PCR reaction product is size separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized with ultraviolet light. Alternatively, the primers may be labeled with a fluorescent dye or a radioactive molecule. Comparison of band intensities between samples allows one to qualitatively estimate the relative starting concentrations of templates amplified, but this method is not quantitative and does not result in determination of the absolute copy number.
A number of quantitative RT-PCR based methods have been described including RNA quantification using PCR and complementary DNA (cDNA) arrays (Shalon et al., Genome Research 6(7):639-45, 1996; Bernard et al., Nucleic Acids Research 24(8):1435-42, 1996), solid-phase mini-sequencing technique, which is based upon a primer extension reaction (U.S. Pat. No. 6,013,431, Suomalainen et al. Mol. Biotechnol. June; 15(2):123-31, 2000), ion-pair high-performance liquid chromatography (Doris et al. J. Chromatogr. A May 8; 806(1):47-60, 1998), and 5′ nuclease assay or real-time RT-PCR (Holland et al. Proc Natl Acad Sci USA 88: 7276-7280, 1991).
It would be useful to develop a method which allows a sensitive and accurate mRNA transcript quantification, can be easily automated and scaled up to accommodate testing of large numbers of sample and overcomes the problems associated with PCR amplification. Such a method would enable diagnosing different pathological conditions, including viruses, bacteria and parasites, as well as different benign and malignant tumors, neurological disorders, heart disease and autoimmune disorders. Such a method would also allow quantifying the transcripts of interest for diagnostic, prognostic and therapeutic purposes, and would ultimately facilitate pharmacogenomic applications. Such a method would also allow screening a large number of agents for effects on gene expression.